eIF4E expression in tumors: its possible role in progression of malignancies

The International Journal of

PERGAMON

The International Journal of Biochemistry & Cell Biology 31 (1999) 59±72

Biochemistry & Cell Biology

Review

eIF4E expression in tumors: its possible role in progression of malignancies Arrigo De Benedetti a, *, Adrian L. Harris b a

Department of Biochemistry and Molecular Biology, Lousiana State University Medical Center, Shreveport, 1501 Kings Highway, P.O. Box 33932, Shreveport LA 71130, USA b University of Oxford, Institute of Molecular Medicine, John Radcli€ Hospital, Headington, Oxford OX3 9DU, UK

Abstract A central issue in the study of neoplastic transformation is to understand how proto-oncogene products deregulate normal processes of cell growth and di€erentiation; an intrinsic aspect of this is to probe the sequence of events leading to altered expression of proto-oncogenes. In the past few years, studies aimed at understanding the regulation and function of protein synthesis initiation factors, eIF4E initially, culminated in the unexpected ®nding that a moderate overexpression of this factor results in dramatic phenotypic changes, including rapid proliferation and malignant transformation. Conversely, the tumorigenic properties of cancer cells can be strongly inhibited by antisense-RNA against eIF4E, or overexpression of the inhibitory proteins: 4E-BPs. Furthermore, eIF4E is elevated in carcinomas of the breast, head and neck (HNSCC) and prostate, but not in typical benign lesions. This is a strong indication that elevated eIF4E expression may mark a critical transition in cancer progression. Establishing a greater protein synthesis output may be a necessary step for cancer cells in order to sustain their rapid proliferation. However, analysis of cells transformed by eIF4E revealed that the synthesis of only a few proteins was greatly enhanced, while synthesis of most was minimally increased. One possible explanation is that eIF4E causes these e€ects by speci®cally increasing the translational eciency of several oncogene transcripts, leading to overexpression of their products. The feasibility of this hypothesis was con®rmed experimentally with the identi®cation of several important products that are speci®cally upregulated in eIF4E-overexpressing cells. These include: c-Myc, cyclin D1 and ODC, which control cycle progression and tumorigenesis; basic ®broblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF), which are powerful promoters of cell growth and angiogenesis. A deeper understanding of the mRNAs that are strongly dependent on excess eIF4E/F for ecient translation will eventually result in fuller understanding of the fundamental role of translational control in di€erent pathophysiological conditions, including malignancy. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: EIF4E oncogene; Tumor vascularization; Cancer progression; Survival factor; Histologic marker

1. Background 1.1. The hierarchy in mRNA translation Protein synthesis plays a fundamental role in

nearly every aspect of metabolism. It also constitutes a critical step in the control of gene expression [1]. The synthesis of each protein ultimately depends on the relative abundance of

* Corresponding author. Tel.: +1-318-675-5668; fax: +1-318-675-5180; e-mail: [email protected]. 1357-2725/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 8 ) 0 0 1 3 2 - 0

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its mRNA and its intrinsic translatability, i.e., the capacity of that particular mRNA to interact with components of the translation initiation machinery. This property of the translation initiation process establishes an order of priorities among the di€erent mRNAs to be translated. Such a hierarchy in protein synthesis is extremely important for gene expression. In eukaryotes, the ¯ow of information from genes to proteins is too slow to accommodate rapid changes in the environment. Eukaryotes compensate for this problem by maintaining a pool of mRNAs that are not immediately utilized. Some of these `translationally repressed' mRNAs may, for instance, encode transcription factors that can be rapidly produced in response to a signal. One example is the key regulator of the general control of amino acid biosynthesis in yeast: the transcription factor Gcn4 [2]. The GCN4 gene is not regulated transcriptionally; instead, depletion of amino acids ultimately results in translational derepression of the GCN4 mRNA. Other examples may include transcripts that encode growth factors which can be rapidly produced under conditions that require cells to re-enter rapid division in response to injury [3]. In metazoans, mRNAs vary over a 100-fold range in their translational eciencies [4]. Moreover, their translation depends on the particular growth conditions of the cell. A theoretical treatment of mRNA competition [5] postulates that the spectrum of translated mRNAs varies with the overall rate of protein synthesis. Weak mRNAs are outcompeted by strong mRNAs when the rate of translation initiation is reduced, as in quiescent cells [6, 7]. A disproportionate number of mRNAs which would be characterized as weak (based on structural features explained later) are those encoding regulators of cell growth. 1.2. List of mRNAs that are known or believed to be translationally repressed Some of these genes are listed below: proto-oncogenes:

c-myc pp60-src lck mos c-fos mdm2 pim-1 growth factors: TGFb-(1, 2, and 3) FGF-2 IL-1b insulin-like-GF (IGF-II) PDGF/c-sis VEGF/VPF growth-promotion genes: ornithine decarboxylase, aminotransferase ribosomal proteins cyclin D1

[8±10] [11] [12] [13±15] [16±19] [20] [21] [22, 23] [24, 25] [26] [27] [28, 22] [29] [30±32], [33] [34, 35] [36, 37].

Many of these genes share the property of being cell-cycle regulated and, in turn, their protein products a€ect cell cycle progression [7, 38]. 1.3. Regulation of protein synthesis Protein synthesis is energetically the most expensive process in the cell and, not surprisingly, translation rates are tightly regulated (reviewed in [39]). In mammals, most of the regulation thus far discovered operates at the level of translation initiation, rather than elongation or termination [40]. The initiation process is comprised of three steps: (1) formation of the 43S complex, composed of a 40S ribosomal subunit and the initiation factors eIF-2, eIF-3, MettRNAi and GTP; (2) formation of the 48S complex containing mRNA, which is mediated by the eIF-4 group of factors; and (3) joining of the 60S subunit to form the complete 80S complex. In most circumstances, the second step is rate limiting and hence, subject to regulation. This step is also a point of discrimination, since one particular mRNA is selected from the untranslated pool and recruited to the ribosomes. As mentioned above, this process is mediated by the eIF4 group of factors, of which eIF4E is the least

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abundant and most likely rate limiting [41, 42]. Evidence supporting this idea was obtained experimentally with the application of antisense RNA technology, since protein synthesis rates and the spectrum of expressed proteins were directly proportional to the level of eIF4E/ 4F [43]. 2. Functions of eIF4E and the paradigm of strong mRNAs eIF4E speci®cally binds to the 7-methylguanosine-containing cap of mRNA in the ®rst step of mRNA recruitment for translation [44]. eIF4E is also a subunit of the eIF4F complex which, in combination with other components of the initiation machinery, unwinds the secondary structure in the 5 0 untranslated region (5 0 UTR) of mRNA. This latter function is critical during `scanning' for exposing and locating the translation start site [45±49]. The low abundance of eIF4E creates a situation of competition among di€erent mRNA species, such that mRNAs with long and highly structured 5 0 UTRs are outcompeted for binding to ribosomes by the strong mRNAs [50]. An analysis of sequence data from 700 vertebrate mRNAs has shown that more than 90% of them contain 5 0 UTRs that are less than 200 nucleotides long and devoid of upstream AUGs, which is characteristic of strong mRNAs [16, 17]. Weak mRNAs contain long G±C-rich 5 0 UTRs, with potential for forming stable secondary structure, and/or upstream AUGs [18, 51, 52]. As indicated above, many such mRNAs code for oncoproteins, regulators of the cell cycle, growth factors and their receptors. It is postulated that by increasing the level/activity of eIF4E the translation of the weak mRNAs would be preferentially elevated. This is based on the kinetic model shown in Fig. 1, which portrays the relative translational eciency of a strong and a weak mRNA as a function of eIF4E level. Translation of strong mRNAs (e.g., those encoding housekeeping proteins) will quickly reach a maximum in the presence of low levels of eIF4E, even in quiescent cells; but weak mRNAs cannot bind to

Fig. 1. mRNA competition for translation initiation. Derived and expanded from the original R* model [5], it was developed here from theoretical considerations and experimental observations [46, 53]. The products of weak mRNAs become selectively increased with higher eIF4E, and can become a substantial fraction of the total spectrum of protein synthesis.

the limiting eIF4E and are translated poorly in these conditions. With higher eIF4E levels, weak mRNAs (e.g., growth regulators) are translated better, enabling the cells to cycle and proliferate. Further increasing eIF4E does not appreciably increase the translation of house-keeping mRNAs, but greatly enhances that of weak mRNAs; the cells are pushed to grow more rapidly and may become neoplastic. An even greater increase in eIF4E could lead to inappropriate expression of key controllers of the cell cycle; the cells then may undergo aberrant mitosis and/or apoptosis, which can lead to further genetic instability and subsequent selection of aggressive survivors. The activity of eIF4E is in turn regulated by various phosphorylation events resulting in increased anity for the cap, tighter association with eIF4G, and release from a set of inhibitory proteins (4E-BPs, reviewed in [37, 54]). This review deals only with changes in the expression of eIF4E, as phosphorylation is discussed elsewhere [55, 56].

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Experimental support for mRNA competition applied to growth regulation was obtained by showing that overexpression of eIF4E speci®cally facilitates the translation of model mRNAs containing excess secondary structure in their 5 0 UTR [25, 57]. Furthermore, overexpression of eIF4E causes malignant transformation and growth deregulation of rodent and human cells [10, 53, 58]. eIF4E also acts as a potent enhancer of transformation in cooperation with v-myc or E1A [59] and Max [10]. Conversely, reducing the level of eIF4E with antisense RNA inhibits the oncogenic and metastatic properties of several oncogenic cell lines [3, 60, 61]. Finally, eIF4E may regulate patterns of di€erentiation, as demonstrated for PC12 cells treated with NGF [62], in regenerating liver [63], and during mesoderm formation in Xenopus embryos [64]. Overexpression of eIF4E can also alter the pattern of lymphokine expression in Th2 lymphocytes, which is characteristic of their state of di€erentiation [65]. Here, we present some of the evidence obtained in several laboratories on the e€ects of overexpressed eIF4E in relation to malignant transformation and then its proposed role and relevance to natural human malignancies. Some emphasis is then placed on the translational regulation of key regulators of the vascular system (e.g., angiogenic inducers, modulators of the in¯ammatory response, proteases involved in remodeling of the interstitial matrix). These factors and processes are essential for progression of tumors, but also for physiological homeostasis. Rapid responses to injury, in¯ammation, anoxia/ ischemia, and tumor angiogenesis would be impaired were they to rely entirely on de novo gene expression. They are all essentially stress responses arising from pathological emergencies. Thus, a mechanism that is based largely on altered translational eciency may have evolved to respond quickly to the imperative of achieving adequate oxygenation. Likewise, cancer cells encounter a need to alter key regulators of protein synthesis, like eIF4E. This, of course, brings up a key question: is eIF4E involved in the initiation or progression of carcinomas? This is a complicated question that cannot be answered as

yet. However, there is now compelling evidence to conclude that elevated eIF4E plays a pivotal role somewhere in the multi-hit process of cancer development. 2.1. Overexpressing eIF4E stimulates division and can transform cells Our understanding of the link of translational control with oncogenesis was initially prompted by two seemingly di€erent observations on the e€ects of eIF4E overexpression in tissue culture. In `normal' cells (NIH3T3, CHO, REF or Th lymphocytes) overexpression of eIF4E resulted in transformation [10, 58, 65]. Similar e€ects were obtained with direct microinjection of the protein [66]. Instead, overexpression of eIF4E in HeLa cells, which are already transformed, resulted in several accelerated divisions culminating in nuclear fragmentation and apoptosis [53]. At the time the relationship between oncogenes, e.g. c-myc, growth stimulation and apoptosis, was unclear (reviewed in [67]). The two observations can now be uni®ed to provide convincing evidence that overexpression of eIF4E produces many of the phenotypic changes that characterize protooncogenes. The idea that a translation factor could transform cells was, at the time, very much out of the mainstream and the mechanism of oncogenesis by eIF4E immediately presented a challenging biological problem. One possibility was that the transforming e€ects of eIF4E are mediated through changes in protein synthesis, as shown in Fig. 2. Overexpression of eIF4E in CHO (or REF) cells resulted in a transformed phenotype, which includes morphologic changes, loss of contact inhibition, shortening of generation time, and growth in soft agar. The level of overexpressed eIF4E was 7-fold, or 20-fold following induction of the inducible promoter with TCDD (Fig. 2A). Consistent with this elevation in eIF4E, the overall rate of protein synthesis was increased 30%± 50%, without and with TCDD, respectively. Analysis of the newly synthesized (pulse-labeled) proteins by SDS/PAGE revealed that, while the

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Fig. 2. Proteins synthesized in CHO cells overexpressing eIF4E. (A) Western blot of eIF4E from extracts of CHO, 4EAla (inactive eIF4E variant), and CHO-4E. (B) Autoradiographic pattern of newly synthesized proteins. Cells were labeled for 1 h with [35 S]methionine. Vector-speci®c bands (encoded by BK-4E) are indicated with arrowheads; these are equally expressed in 4EAla and CHO-4E cells. Proteins that are uniquely elevated in CHO-4E cells are marked with horizontal lines at the right. + indicates induction of the promoter controlling eIF4E with TCDD (tetrachloro-dibenzo-dioxin) (reproduced from [10]).

synthesis of most proteins was only modestly increased, the rate of synthesis of several polypeptides was greatly enhanced [10]. This result is in agreement with the theoretical treatment of mRNA competition for translation described above. Several of these bands (Fig. 2B) are presumably the product of pre-existing, translationally repressed mRNAs. A systematic identi®cation and cloning of the cDNAs encoding these products is a major goal of our laboratories. Meanwhile, in an attempt to identify some of these products, we carried out western blots for several important regulators of cell growth. These were chosen based on similarities with phenotypic e€ects produced by certain proto-oncogenes, on the recognition of speci®c structural features in the 5 0 UTR of their transcripts, and on the size of their protein products.

2.2. Elevated eIF4E results in overexpression of c-Myc, cyclin D1, and ODC c-myc, our ®rst candidate, was chosen because its expression is believed to be translationally regulated [8], and because of its known oncogenic properties [68]. We found that c-Myc was increased 5-fold in CHO-4E (and in CREF-4E) with respect to CHO or CREF, which is comparable to the elevation found in neoplasms where c-myc involvement is suspected (Fig. 3A) [10]. It should be noted that further induction of the vector promoter controlling eIF4E with TCDD does not result in additional increase in Myc expression. This is due to a complex pattern of autoregulation by c-myc (see [10] for a fuller description on this topic). Other studies have revealed that eIF4E enhances the expression of cyclin D1 and polya-

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Fig. 3. Elevated c-Myc and FGF-2 expression in CHO-4E cells. (A) Western blot of c-Myc. The proteins from the indicated cells were probed with anti-c-Myc antiserum. Cells treated with TCDD for 48 h to increase the expression of eIF4E are indicated with a +. The band marked Myc1 is the CUG1-initiated form [69]. The double arrow marks Myc2 (AUG-form, 2phosphorylated). (B) Western blot of FGF-2. 30 mg of protein from each sample were separated on a 15% polyacrylamide-SDS gel, transferred to a membrane and probed with FGF-2 antiserum. (C) Immunoprecipitation (IPPT) of FGF-2. For IPPT, the cells were labeled for 1 h with [35 S]methionine, and homogenized in RIPA. 150,000 cpm from each sample were used for IPPT, with 3 mg of anti-FGF-2 and protein G beads. The di€erent (CUGs and AUG) forms of FGF-2 and their approximate sizes are indicated [3, 24, 70] (reproduced from [25]).

mine enzymes [31, 33, 71, 72]. Cyclin D1 and polyamines are required for entry into S phase and their overexpression has been linked to transformation. 2.2.1. FGF-2 Further progress on the oncogenic properties of eIF4E came with the observation that conditioned medium from cells transformed with eIF4E is strongly mitogenic, in particular to vascular endothelial cells. Basic ®broblast growth factor (FGF-2) belongs to a family of multifunctional molecules that act as potent mitogens for a variety of meso-ectodermal lineages [73±75]. FGF-2 is also a powerful angiogenic factor, involved in both normal and pathologic processes such as wound repair [76] and tumor vascularization [77]. The importance of FGF-2 to breast cancer became apparent with the discovery that the mouse mammary tumor virus (MMTV) integrates preferentially at a genetic locus (int-2) that encodes FGF [78]. A high level of FGF-2 in breast carcinomas is considered to be a poor prognostic determinant (for a review see [74, 75]).

Although an increase in FGF-2 is prevalent in invasive breast carcinomas, ampli®cation at the gene or mRNA level is found in only 10±15% of the cases [79], suggesting that the mechanism of upregulation is post-transcriptional [80]. As shown in Fig. 3, we found a dramatic (>40-fold) increase in FGF-2 expression by immunoprecipitation and western-blot analyses of extracts from CHO-4E (or CREF-4E) vs. control cells [25]. We further demonstrated that the increase was at the level of translation by in vivo and in vitro experiments, and this was also re¯ected in a panel of breast cancer biopsies [3, 70]. 2.2.2. VEGF/VPF Vascular endothelial growth factor/vascular permeability factor is perhaps the most speci®c cytokine for vascular endothelia [81]. As mentioned above, conditioned medium from CHO4E was strongly mitogenic for endothelial cultures (HUVEC). Clearance with antibodies against FGF-2 only partially compromised this activity, indicating that additional factor(s) may be overproduced. VEGF was the next most logi-

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cal candidate and, in fact, it was overexpressed over 100-fold [29]. The increase was almost exclusively translational since in CHO-4E, VEGF mRNA partitioned primarily to the heavy-polysome region, in contrast to its distribution on the small polysomes in control cells. The regulation of VEGF expression, particularly by hypoxia, is attributed to both transcriptional and post-transcriptional mechanisms [82]. We should point out that translation of the same mRNAs (FGF-2, VEGF and c-myc) that we have found to be enhanced in eIF4E-overexpressing cells, were also recently reported to contain IRESs [80, 83, 84]. As such, their translation is expected to occur even in presence of low eIF4E (i.e., cap-independent). While this is certainly a possibility, our results on the translation of these mRNAs are not consistent with a true IRES-driven mechanism of translation, but rather with internal repositioning, also called shunting or jumping [25, 85]. For the human cmyc, we have proposed a model for translation initiation where a ribosome enters the mRNA from the 5 0 UTR (cap-dependent) and scans until it reaches the IRPE (internal repositioning element). The ribosome then pauses and will either resume scanning through the IRPE secondary structure (with the help of eIF4E/F), or it is repositioned at the internal AUG. This model could account for translation of the CUG and AUG-initiated isoforms of myc and FGF-2. Clearly, isolated elements form the 5 0 UTR of these mRNAs should stimulate internal initiation in bicistronic constructs, given the similar function that IRES and IRPE must perform. The recent reports of an IRES in segments from these mRNA (in the context of bicistronic constructs) do not prove IRES activity in the natural context. In fact, the existence of a true IRES in the 5 0 UTR of c-myc was contradicted by its failure to direct translation of a circular transcript, in contrast to hsp70 mRNA [85]. In other words, while the evidence suggests that synthesis of Myc probably occurs by internal-initiation, it does not occur by a mechanism involving internal entry of ribosomes, but rather by ribosome repositioning (shunting). Thus, it still remains to be demonstrated that translation of these mRNAs can

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occur under conditions of limiting eIF4E, while it is clear that their translation is specially enhanced by excess eIF4E. 2.2.3. Metalloproteases, cathepsin, stromelysin, etc. Various types of collagenases/®brinolytic enzymes are critical for remodeling of the stromal layers surrounding the tumors: for subsequent recruitment of supporting vasculature; for development of a complex in¯ammatory response against the tumor cells (that has both bene®cial and detrimental e€ects); and for tumor metastasis. Reduction of eIF4E with antisense RNA inhibits the expression of the 92 kDa collagenase type IV in Ras-transformed CREF cells, possibly inhibiting their metastatic properties [61]. We don't know the mechanism for this reduction since the mRNA encoding this metalloprotease appears rather unremarkable, structurally. 2.3. eIF4E is ubiquitously overexpressed in human carcinomas A critical issue that needed to be resolved is whether eIF-4E is a bona ®de proto-oncogene. Despite the observations that eIF4E can transform cell lines, information about its expression in naturally occurring malignancies, or even its relevance to human cancer, was lacking. We now know that overexpression of eIF4E is rather ubiquitous in solid tumors and malignant cell lines [86, 105]. 2.3.1. Breast cancer Following an initial screen of several types of carcinomas by western blotting, we found that the highest and most reproducible eIF4E elevation was in breast cancer. In particular, the basal level of eIF4E in normal breast is lower than in many other tissues, making the abnormal eIF4E expression in carcinomas very obvious [3, 87, 88]. These results exceeded our expectations, and they rather suggest that the eIF4E elevation is a necessary transition in the progression of breast cancer instead of a direct cause. We have now analyzed a large enough number of breast carcinomas for the level of

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overexpressed eIF4E to be able to make statistically signi®cant conclusions for its role as an independent prognostic indicator [89, 90]. The mechanism of eIF4E overexpression is still under investigation, but initial work suggests that in some cases it is primarily transcriptional. For example, we have found increased eIF4E expression during hypoxic conditions ± hypoxia is an inescapable condition for progression of primary tumors beyond a few millimeters (see below). In other cases it re¯ects gene ampli®cation [91]. Interestingly, ampli®cation of the eIF4G gene, the partner of eIF4E, was reported as a frequent event in squamous cell lung carcinomas [92]. 2.3.2. HNSCC Head and neck squamous cell carcinomas (HNSCC) have a high local recurrence rate due to incomplete tumor resection. In fact, the high lethality of this type of cancer is not due to metastasis, but to local recurrence in most cases. While obtaining clear margins in breast cancer does not usually present a surgical problem, when operating on head and neck lesions every millimeter counts. Further excision can result in additional dis®gurement, loss of functionality, and severe hemorrhage. Incomplete excision will almost invariably result in rapid recurrence and death from tumor burden. Margins are determined during surgery by histopathologic analysis on frozen sections. Unfortunately, benign/atypical dysplasia and ulcerations are very common in the oral cavity of these patients, after years of excess smoking and drinking. Even the best pathologists have great diculty in distinguishing between malignant and preneoplastic lesion. We postulated that genetic and molecular changes precede the gross histologic alterations that qualify such lesions as cancerous. Thus, tumor markers may improve the reliability of pathological examination, and this presented our ®rst opportunity of using eIF4E to improve the determination of clear margins. This was possible because eIF4E is not overexpressed in benign lesions, such as polyps and leukoplakia, but it is clearly elevated in HNSCC. We don't know the limit of sensitivity of our assay (we don't know

how few cancer cells present at the margins can give a positive signal by western blot), but it was sucient to predict local recurrence in two-thirds of the patients who have recurred within 2 years [70]. In a recent retrospective study, 66% of patients with elevated eIF4E in histologically `tumor-free' margins have recurred and died within 3 years (n = 18). Only one in the group of patients who had clear margins also by eIF4E determination (n = 13) had a local recurrence [93]. 2.4. The advantage for tumor cells and the pathophysiology As introduced above, translational control exerted by low eIF4E/F is critical for the appropriate repression of gene expression needed to maintain organ/tissue di€erentiation. Conversely, there seems to be an obvious need for a mechanism that is based largely on altered translational eciency to respond quickly to pathological emergencies. One clear-cut example for this concept is the process of neovascularization. The process of neovascularization is the perennial capacity of the organism to provide new capillary sprouts to tissues in need. This re¯ects both physiological situations, such as growth of endometrial tissue during the reproductive cycle or placental development, and pathological conditions like ischemia/hypoxia, vascular retinopathy and tumor angiogenesis. The process of neovascularization is complex and essentially involves: (i) degradation of the vascular basement membrane and the ®brin interstitial matrix; (ii) migration of endothelial cells; (iii) rapid proliferation of endothelial cells; and (iv) formation of new capillary tubules and deposit of a new basement membrane [76]. The entire process must be remarkably fast to minimize blood and ¯uid loss and yet, stop as soon as the area in need is adequately vascularized. Rapid responses to injury, in¯ammation, or tumor angiogenesis would be impaired were they to rely entirely on de novo gene expression. Instead, a mechanism that is based largely on altered translational eciency may have evolved to respond quickly to the imperative of achieving adequate oxygenation. Elevated levels of eIF4E are present in most

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invasive ductal carcinomas, and this may be essential for cancer progression. We propose that upregulating eIF4E/F is a pre-requisite for escaping the partially anoxic environment of con®ned cancerous lesions and for vascularization and metastasis of the primary tumor [3]. Elevated eIF4E correlates with increased expression of FGF-2 [3] and VEGF [88] in tumor biopsies. Furthermore, the level of VEGF mRNA correlates strongly with the level of eIF4E mRNA ( p = 0.0002) [88]. As mentioned above, these are probably the two most powerful mitogens for vascular endothelia and they work synergistically. A role for elevated eIF4E in the angiogenic phase and progression of breast carcinomas was obtained with experiments in which the level of eIF4E was suppressed with antisense RNA technology. When the level of eIF4E was reduced to near normal level in MDA-435 cells (human breast cancer) [3] or in PC-3 (human prostate cancer) [94] their angiogenic and tumorigenic properties were crippled. The viability and tissue culture properties, including the capacity to grow in soft agar, of 435AS and PC3AS (i.e., antisense RNA to eIF4E) cells were otherwise una€ected. 2.5. eIF4E and protection from apoptosis The balance between growth, quiescence, and programmed death (apoptosis) is a delicate process required for the homeostasis of the organism and all the way down to individual cells. It is accomplished by the orchestrated expression of survival (e.g., Bcl-2) and pro-apoptotic factors, like TNF and FasL [95]. Given the rapid growth of tumor cells, one concept that is sometimes counterintuitive is the fact that cancer cells are often one step away from being dead. For instance, the loss of functional DNA repair genes, which is intimately linked to mechanisms of carcinogenesis and genetic instability [96], is also necessarily linked to the accumulation of lethal mutations and increased sensitivity to chemotherapy by individual cancer cells. To counter this, cancer cells increase their o€spring production and o€set the apoptotic balance by altering the expression of survival factors. It was recently found that eIF4E can act as a potent

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Fig. 4. Sensitivity of parental MDA435 (*) and 435AS (Q) cells to the cytotoxic peptide Bleomycin. Average of three replicated experiments with standard deviation.

survival factor for serum-deprived cells, or for those with deregulated expression of Myc [97]. Conversely, we found that the 435AS cells had increased apoptotic index in serum-deprived medium and were very sensitive to the radiomimetic drug bleomycin (Fig. 4). This suggests that tumor cells with high eIF4E may be quite resistant to chemotherapy, even before development of multidrug resistance. 2.6. eIF4E as a new therapeutic target The results shown in Fig. 4 also suggest that eIF4E could be used as a therapeutic target. Not only did the suppression of eIF4E result in decreased tumorigenic capacity, but also the cells became much more sensitive to a chemotherapeutic agent that mimics the e€ects of radiation. Although caution must always be used when extrapolating results in tissue culture to their application in the clinics, it seems logical to begin thinking of eIF4E as a possible target for combination therapy. Another reasonable approach is to target the pathway(s) that lead to phosphorylation and inactivation of the inhibitors 4E-BPs (reviewed in [54]). Indeed, many known inhibitors of the signal transduction pathway leading to cell proliferation (Rapamycin, Wortmannin, LY294002, commonly used as immunosupressants) act on mTOR/FRAP, that can directly

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phosphorylate 4E-BP. Inhibition of mTOR by these molecules prevents the phosphorylation of 4E-BP by mitogenic signals, thereby sequestering eIF4E from the pool available for translation initiation. An indication that such approach is feasible was given by the fact that overexpression of 4EBP1 could inhibit the oncogenic properties of src-transformed cells [72, 98]. 3. Concluding remarks In conclusion, we found that eIF4E is widely overexpressed in solid tumors, while its oncogenic potential is already well-established in tissue culture. Establishing greater protein synthesis outputs may be a necessary development which cancer cells must accomplish in order to sustain their rapid proliferation. Nonetheless, elevating eIF4E appears to have a speci®c e€ect on the pattern of protein synthesis, rather than a generalized increase. However, when identi®ed, several of these products turned out to be factors that are of signi®cant importance to cancer progression. Future progress will come from a better understanding of the mechanism of eIF4E upregulation and from identi®cation of mRNAs that require excess eIF4E for ecient translation. Future directions of investigation clearly will require an analysis of the structure and regulation of the genes encoding the eIF4 group of translation factors. For instance, the promoter of eIF4E is regulated by c-Myc [99], a cell-cycle regulator that is found elevated in a large proportion of human cancers. However, the regulation of eIF4E expression is likely to be complex. The gene encoding eIF4E was only recently cloned in its entirety [100], and the functional elements are not fully elucidated. Furthermore, the eIF4E gene resides on 4q2122 [101], a region of the chromosome that is frequently ampli®ed in prostate cancer of African Americans [102]. Interestingly, trisomy of chromosome 4 with double minutes of chromosome 8, involving the c-myc locus, is one of the most frequent rearrangements found in AML [103]. Finally, a full structural elucidation

and cloning of the eIF4 genes is a high priority, as it is possible that under di€erent physiological demands the composition of the eIF4F complex may change, resulting in a di€erent spectrum of protein synthesis. For instance, a fully functional human homologue of eIF4G-I was recently cloned [104]. Two eIF4G genes are also found in yeast and plants, suggesting that this may be a universal feature in eukaryotes. It is already established that eIF4G acts as an interface for the association of other initiation factors along the mRNA 5 0 UTR. An attractive idea is that the recruitment and assembly of the eIF4F complex may involve a modular selection process under di€erent protein synthesis needs, similar to the assembly of s factors in gene expression of prokaryotes.

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